The present invention relates to the production of recombinant proteins in bacterial host cells on a manufacturing scale.
Currently, production of recombinant proteins in bacterial hosts, in particular Escherichia coli, mostly uses plasmid-based expression systems. Since these systems provide high gene dosage and are well established, they have become widely accepted, also because the available cloning protocols are simple to handle.
Plasmid-based expression is characterized by plasmid copy numbers up to several hundred per cell (Baneyx, 1999). Expression plasmids usually carry the gene of interest under the control of a promoter, an origin of replication (ori) and a marker gene for selection of plasmid-carrying clones. In addition, coding or non-coding or non-functional backbone sequences are frequently present on said plasmids (i.e. vectors). The presence of plasmids and the corresponding replication mechanism alter the metabolism of the host cell (Diaz-Rizzi and Hernandez, 2000) and impose a high metabolic burden on the cells, thereby limiting their resources for recombinant protein production. In addition, the application of strong promoters in combination with high gene dosage triggers a rate of recombinant protein formation that is usually too high for the host cell to cope with and may therefore lead to a quick and irreversible breakdown of the cell metabolism. Consequently, the host cell's potential cannot be fully exploited in plasmid-based systems, resulting in low yield and quality of the recombinant protein. Thus, one of the major drawbacks of plasmid-based expression systems may be attributed to the increased demand for nutrients and energy that is required for plasmid replication and maintenance.
Another typical phenomenon in plasmid-based systems is the change of plasmid copy number in the course of cultivation. Recombinant protein production is accompanied, at high expression rates, with starvation and cellular stress that lead to increased pools of uncharged tRNAs. This leads to an interference with the control mechanism of plasmid copy number (PCN). Consequently, PCN increases rapidly and causes a breakdown of the cultivation process (so-called “run-away effect”). The run-away phenomenon of ColE1 type plasmids after induction of recombinant gene expression can lead to a strong increase of gene dosage (Grabherr et al., 2002).
Segregational instability, (i.e. the formation of plasmid free host cells) and structural instability (i.e. mutations in plasmid sequence) are further problems often seen in plasmid-based systems. During cell division, cells may lose the plasmid and, consequently, also the gene of interest. Such loss of plasmid depends on several external factors and increases with the number of cell divisions (generations). This means that plasmid-based fermentations are limited with regard to the number of generations or cell doublings (Summers, 1991).
Overall, due to these properties of plasmid-based expression systems, there is a limited yield of recombinant protein and a reduced controllability of process operation and process economics. Nevertheless, due to lack of more efficient alternatives, plasmid-based bacterial expression systems became state of the art for production and isolation of heterologous recombinant proteins on a manufacturing scale.
Therefore, as an alternative to plasmid-based expression, genome-based expression systems have been explored. A well-known and widely-applied example for a heterologous protein that is chromosomally expressed in E. coli, is the RNA polymerase of the T7 phage, which serves the purpose of plasmid-based transcription of a plasmid-based gene of interest. This system, which is originally described in U.S. Pat. No. 4,952,496, is based on non-site specific integration, and renders the resulting bacterial strains (e.g. E. coli BL21(DE3) or HMS174(DE3)) as lysogens. To prevent potential cell lysis followed by undesired phage release, the T7 polymerase gene was integrated into the chromosome without creating phage lysogen, i.e. it was inserted by homologous recombination (WO 2003/050240). Recently, integration of T7 RNA polymerase gene into the genome of Corynebacterium acetoacidophilum has been described, again for the purpose of plasmid-based expression of recombinant proteins (US 2006/0003404).
Other methods for genomic integration of nucleic acid sequences—in which recombination is mediated by the Red recombinase function of the phage λ (Murphy, 1998) or the RecE/RecT recombinase function of the Rac prophage (Zhang et al., 1998)—have been suggested for protein expression studies, sequence insertions (e.g. of restriction sites, site-specific recombinase target sites, protein tags, functional genes, promoters), deletions and substitutions (Muyrers et al., 2000).
WO 2001/18222 describes utilization of a chromosomal integration method based on the Saccharomyces cerevisiae FLP (flippase)/FRT (flippase recognition target) recombination system (Pósfai et al., 1994) for insertion of ethanol pathway genes from Zymomonas mobilis downstream of chromosomal promoters of E. coli in order to confer ethanologenic properties to that host. The FLP system was used for precisely removing sequences (markers and replicons) after chromosomal integration of circular vectors (Martinez-Morales et al., 1999).
A method developed by Datsenko and Wanner (2000) for insertion of linear DNA fragments with short homology sequences, utilizing the λ Red technology in combination with the FLP-based marker excision strategy, has been applied for the insertion of an antibiotic resistance gene for chromosomal gene replacement (Murphy, 1998) or for gene disruption (Datsenko and Wanner, 2000). Also, this method has been suggested for overexpression of tagged homologous E. coli proteins by the chromosomal insertion of a marker, a promoter and a His-tag prior to (i.e. upstream of) said proteins (Jain, 2005).
Genome-based expression was also suggested for integration of a repressor molecule into the chromosome of E. coli to establish a host/vector selection system for propagation of plasmids without an antibiotic resistance marker (WO 2006/029985). In the context of that selection system, the genomic insertion of a reporter protein (green fluorescent protein) was used as a model system to demonstrate a plasmid-derived RNAI/II antisense reaction (Pfaffenzeller et al., 2006). In a similar way, Zhou et al., (2004) described the integration of a circular vector carrying green fluorescent protein as a reporter molecule.
In WO 1996/40722, a method is described that makes use of integration of a circular vector (so-called “circular chromosomal transfer DNA”, CTD) including a selectable marker into the bacterial chromosome (i.e. at the attB site of E. coli). In that method, by using duplicate DNA sequences flanking the selection marker, amplification of the chromosomal gene dosage was achieved. Thereby, the obtained chromosomal gene dosage was approximately 15-40 copies per cell, which is similar to those achieved by commonly used plasmid vectors. Cultivation of clones containing chromosomal transfer DNA integrated into the bacterial genome resulted in levels of recombinant proteins similar to those obtained by plasmid-based systems (Olson et al., 1998). This method requires in vitro ligation of CTD and is, with regard to integration, limited to the attB site.